CN101816799A - Gene vaccine of sperm-specific cationic channel protein CatSper1 and preparation method thereof - Google Patents
Gene vaccine of sperm-specific cationic channel protein CatSper1 and preparation method thereof Download PDFInfo
- Publication number
- CN101816799A CN101816799A CN201010143964A CN201010143964A CN101816799A CN 101816799 A CN101816799 A CN 101816799A CN 201010143964 A CN201010143964 A CN 201010143964A CN 201010143964 A CN201010143964 A CN 201010143964A CN 101816799 A CN101816799 A CN 101816799A
- Authority
- CN
- China
- Prior art keywords
- catsper1
- baff
- vaccine
- full
- length cdna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a vaccine, in particular to a gene vaccine of sperm-specific cationic channel protein CatSper1. The vaccine is an immune-stimulating complex type vaccine composed of CatSper1-BAFF digenic recombinant eukaryotic vector, saponin Quil A, cholesterol and lecithin; and the vaccine can inhibit the movement of sperms, perform specific reduction of the average farrowing rate and number born of BALB/c mouse and have good application prospect in the immunological contraception field. The invention also relates to a preparation method of the vaccine which has simple operation and low cost.
Description
Technical field
The present invention relates to a kind of vaccine, the particularly gene vaccine of sperm-specific cationic channel protein CatSper 1 also relates to the preparation method of this vaccine.
Background technology
Represented a kind of on the make contraception approach based on the vaccine development of sperm antigen.The application of sperm antigen in pregnancy vaccine exploitation is its sperm-specific, participates in human fertility, and can be enough to the immunne response that can stop being fertilized local generation of reproductive tract.
CatSper1 is the distinctive a kind of cationic channel protein of sperm, is expressed in testis and epididymis tissue specifically, mainly is positioned the principal piece of sperm tail, for the sperm superactivation important regulatory role is arranged.Genetic knock-out experiment shows that the sperm motility power of CatSper1 gene delection mice reduces greatly, can not pass the intact ovum of zona pellucida, and the forfeiture fertility.
The B cell-stimulating factor (BAFF) is the B cell survival and the sophisticated essential factor, is tumor necrosis factor (TNF) family member.It can strengthen B cell, cd4 t cell, NK cell activity, and the immunne response of body is strengthened; Also can be used as the costimulating factor of t cell activation, stimulate T emiocytosis interferon-(IFN-γ) and interleukin-2 (IL-2), raise CD25 (IL-2 receptor alpha chain) and express, and promote T hyperplasia in IL-2 dependence mode.
Immunostimulating complex (ISCOM) is the spherical cage shape granule that the spontaneous a kind of diameter that forms is 30～40nm after being mixed by antigen, saponin Quil A, cholesterol and phospholipid, dual-use function with adjuvant and antigen presentation can increase substantially immunogenicity of antigens.Bibliographical information; be that the inductive neutralizing antibody level of dna vaccination that makes of adjuvant and immune protective efficiency are apparently higher than naked DNA with ISCOM; possible mechanism is can avoid the degraded of nucleic acid in vivo enzyme through the DNA of ISCOM parcel, thereby guarantees this DNA continuous expression in body.In addition, the architectural characteristic of ISCOM makes it be easier to settle down in lymphoid tissue, thereby helps engulfing of antigen presenting cell.
Summary of the invention
In view of this, one of purpose of the present invention is to provide a kind of gene vaccine of sperm-specific cationic channel protein CatSper 1; Two of purpose is to provide the preparation method of the gene vaccine of described sperm-specific cationic channel protein CatSper 1.
For achieving the above object, the present invention adopts following technical scheme:
1, the gene vaccine of sperm-specific cationic channel protein CatSper 1, the ISCOM type vaccine that this vaccine is made up of CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector, saponin Quil A, cholesterol and lecithin.
Further, described CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector is that IRES upstream and downstream at the double gene coexpression eukaryotic vector that contains internal ribosome entry site IRES inserts CatSper1 full-length cDNA and BAFF full-length cDNA respectively;
Further, the mass ratio of described CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector, saponin Quil A, cholesterol and lecithin is 5: 10: 1: 1.
2, the preparation method of the gene vaccine of described sperm-specific cationic channel protein CatSper 1 may further comprise the steps:
The structure of a, CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector: the IRES upstream of the CatSper1 full-length cDNA being inserted the double gene coexpression eukaryotic vector that contains IRES, the IRES downstream that B cell-stimulating factor full-length cDNA is inserted this eukaryotic vector promptly gets CatSper1 and B cell-stimulating factor double gene coexpression reorganization eukaryotic vector again;
The preparation of b, ISCOM: is after PBS dissolves with CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector with phosphate buffer, add saponin Quil A, add cholesterol and lecithin again, make and contain CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector 0.5mg, saponin Quil A1mg, cholesterol 0.1mg and lecithin 0.1mg in every ml soln, ice-bath ultrasonic is handled and is made abundant mixing of solution and emulsifying, reuse PBS dialysis, the little floccule of gained is the ISCOM of formation, also is the gene vaccine of CatSper1.
Further, the concrete grammar of described step a is:
A1, CatSper1 Full Length cDNA Cloning: extract people's mature sperm cell total rna, reverse transcription obtains total cDNA, be template with this total cDNA again, with sequence shown in SEQ ID No.3 and the SEQ ID No.4 is the upstream and downstream primer, and the pcr amplification two ends have the CatSper1 full-length cDNA of Pst I and EcoR I restriction enzyme site respectively, and the PCR condition is: 94 ℃ of pre-degeneration 3 minutes, 1 minute, 58 ℃ annealing of 94 ℃ of degeneration are 1 minute then, 72 ℃ were extended 1 minute, totally 30 circulations, and last 72 ℃ were extended 7 minutes; The purified rear clone of PCR product is gone into the pT-Easy carrier, gets recombinant vector pT-CatSper1;
A2, BAFF Full Length cDNA Cloning: extract the total RNA of people's spleen tissue, reverse transcription obtains total cDNA, be template with this total cDNA again, with sequence shown in SEQ ID No.5 and the SEQ ID No.6 is the upstream and downstream primer, the pcr amplification two ends have the B cell-stimulating factor full-length cDNA of BamH I and Sal I restriction enzyme site respectively, and the PCR condition is: 94 ℃ of pre-degeneration 5 minutes; 94 ℃ of degeneration are 50 seconds then, 58 ℃ of annealing 50 seconds, and 72 ℃ were extended totally 30 circulations 2 minutes; Last 72 ℃ were extended 10 minutes; The purified rear clone of PCR product is gone into the pT-Easy carrier, gets recombinant vector pT-BAFF;
The structure of a3, reorganization eukaryotic vector: in step a1 gained recombinant vector pT-CatSper1, go out the CatSper1 full-length cDNA with Pst I and EcoR I double digestion, purified back be connected with the eukaryotic vector pStar of EcoR I double digestion with Pst I equally, must recombinant vector pStar-CatSper1; In step b gained recombinant vector pT-BAFF, go out the BAFF full-length cDNA again with BamH I and Sal I double digestion, purified back be connected with the recombinant vector pStar-CatSper1 of Sal I double digestion with BamH I equally, promptly get CatSper1 and the BAFF double gene coexpression eukaryotic vector pStar-CatSper1-IRES-BAFF that recombinates.
Beneficial effect of the present invention is: the gene vaccine of sperm-specific cationic channel protein CatSper 1 of the present invention can suppress the motion of sperm, specificity reduces the average farrowing rate and the litter size of BALB/c mouse, and preparation method is simple, with low cost, has a good application prospect in the immunological contraception field.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 is the agarose gel electrophoresis result of pcr amplification CatSper1 full-length cDNA;
Fig. 2 is the agarose gel electrophoresis result of pcr amplification BAFF full-length cDNA;
Fig. 3 detects CatSper1 for immunohistochemistry;
Fig. 4 detects CatSper1 for immunofluorescence;
Fig. 5 detects for the percentage rate of motion sperm;
Fig. 6 is the average litter size of female BALB/c mouse in the breeding pairing experiment;
Fig. 7 is the average farrowing rate of female BALB/c mouse in the breeding pairing experiment.
The specific embodiment
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.
One, the preparation of CatSper1 gene vaccine
1, the structure of CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector
(1) CatSper1 Full Length cDNA Cloning
According to the GenBank accession number is the CatSper1 gene order of NM_053054, designs and synthesizes following primer has restriction enzyme site with the pcr amplification two ends CatSper1 full-length cDNA: forward primer F1:5 '-aat
CtgcagAtggatcaaaactcagtgcct-3 ' (SEQ ID No.3), underscore partly is a Pst I restriction enzyme site; Downstream primer R1:5 ' tac
GaattcTcaattcctgaagtcctcttctc-3 ' (SEQ ID No.4), underscore partly is an EcoR I restriction enzyme site; From the healthy male volunteers seminal fluid, isolate mature sperm, cultivate with RPMI 1640 culture medium are conventional, collecting cell, PBS washing 3 times is extracted the total RNA of spermatid with the total RNA extraction reagent box; With the total RNA reverse transcription of gained sperm is cDNA, be template, adopt upstream and downstream primers F 1 and R1 to carry out pcr amplification with this cDNA again, the PCR condition is: 94 ℃ of pre-degeneration 3 minutes, 1 minute, 58 ℃ annealing of 94 ℃ of degeneration are 1 minute then, 72 ℃ were extended 1 minute, totally 30 circulations, last 72 ℃ were extended 7 minutes; The PCR product is after agarose gel electrophoresis evaluation, gel recovery test kit are cut glue recovery purification, be connected with pT Easy carrier, connect product transformed into escherichia coli JM109 competent cell, with the LB plate screening positive colony that contains ampicillin, extract plasmid, entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's sequence verification, will insert the positive colony plasmid called after recombinant vector pT-CaSper1 of CaSper1 full length cDNA sequence (SEQ ID No.1);
The agarose gel electrophoresis result of PCR product as shown in Figure 1, wherein the M swimming lane is a dna molecular amount standard, 1 swimming lane is the PCR product, visible PCR product is the single specificity band at about 2300bp place, conforms to the purpose clip size.
(2) BAFF Full Length cDNA Cloning
According to the GenBank accession number is the BAFF gene order of NM_006573, designs and synthesizes following primer has restriction enzyme site with the pcr amplification two ends BAFF full-length cDNA: forward primer F2:5 '-gatca
GgatccAtggatgactccacagaaagg-3 ' (SEQ ID No.5), underscore partly is a BamH I restriction enzyme site; Downstream primer R2:5 ' acgc
GtcgacTcacagcagtttcaatgcac-3 ' (SEQ ID No.6), underscore partly is a Sal I restriction enzyme site; Press Trizol test kit description and extract the total RNA of people's spleen tissue, reverse transcription obtains total cDNA, is template with this total cDNA again, adopts upstream and downstream primers F 2 and R2 to carry out pcr amplification, and the PCR condition is: 94 ℃ of pre-degeneration 5 minutes; 94 ℃ of degeneration are 50 seconds then, 58 ℃ of annealing 50 seconds, and 72 ℃ were extended totally 30 circulations 2 minutes; Last 72 ℃ were extended 10 minutes.The PCR product is after agarose gel electrophoresis evaluation, gel recovery test kit are cut glue recovery purification, be connected with the pT-Easy carrier, connect product transformed into escherichia coli DH5 α competent cell, with the LB plate screening positive colony that contains ampicillin, extract plasmid, entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's sequence verification, will insert the positive colony plasmid called after recombinant vector pT-BAFF of BAFF full length cDNA sequence (SEQ ID No.2).
The agarose gel electrophoresis result of PCR product as shown in Figure 2, wherein the M swimming lane is a dna molecular amount standard, 1 swimming lane is the PCR product, visible PCR product is the single specificity band at about 800bp place, conforms to the purpose clip size.
(3) structure of CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector
According to the designed restriction enzyme site of CatSper1 and BAFF full-length cDNA two ends, earlier the CaSper1 full-length cDNA is inserted the IRES upstream of the double gene coexpression eukaryotic vector pStar that contains internal ribosome entry site IRES, again the BAFF full-length cDNA is inserted the IRES downstream of pStar carrier.Concrete grammar is: earlier with recombinant vector pT-CaSper1 Pst I and EcoR I double digestion, the double digestion product is after gel recovery test kit is cut glue recovery purification, with be connected with the eukaryotic vector pStar of EcoR I double digestion through Pst I equally, connect product transformed into escherichia coli DH5 α competent cell, with the LB plate screening positive colony that contains ampicillin, extract plasmid, Pst I and EcoR I double digestion are identified, with positive colony plasmid called after recombinant vector pStar-CatSper1; Again with recombinant vector pT-BAFF BamH I and Sal I double digestion, the double digestion product is after gel recovery test kit is cut glue recovery purification, with be connected with the recombinant vector pStar-CatSper1 of Sal I double digestion through BamH I equally, connect product transformed into escherichia coli DH5 α competent cell, with the LB plate screening positive colony that contains ampicillin, extract plasmid, BamH I and Sal I double digestion are identified, with positive colony plasmid called after CaSper1 and BAFF double gene coexpression reorganization eukaryotic vector pStar-CatSper1-IRES-BAFF.
2, the preparation of ISCOM
Is after PBS dissolves with CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector pStar-CatSper1-IRES-BAFF with phosphate buffer, add saponin Quil A, add cholesterol and lecithin again, make and contain CaSper1 and BAFF double gene coexpression reorganization eukaryotic vector 0.5mg, saponin Quil A 1mg, cholesterol 0.1mg and lecithin 0.1mg in every ml soln, ice-bath ultrasonic is handled and is made abundant mixing of solution and emulsifying, reuse PBS dialysis, the little floccule of gained is the ISCOM of formation, also is the CatSper1 gene vaccine.
Two, immunity and detection in the body of CaSper1 gene vaccine
Male BALB/c mouse is divided into experimental group and blank group at random, 5 every group; Experimental group is an immunogen with CaSper1 gene vaccine of the present invention, and the blank group is immunogen with PBS; Two groups of mices are pressed the capable peritoneal immunity of dosage of 100 μ L, every 2 week immunity 1 time, continuous immunity 3 times; The 14th day tail vein blood after the last immunity, separation of serum is measured serum antibody titer with indirect elisa method.
(1) immunohistochemistry detects CaSper1
Get mouse testis tissue (0.5cm * 0.5cm * 0.5cm) put in the neutral formalin solution, fix 12 hours for 4 ℃, dehydration back paraffin embedding, section (5mm), the row immunohistochemistry detects: the wax section is dewaxed to water, with volume fraction is 3% hydrogenperoxide steam generator incubated at room 5～10 minutes, distilled water flushing, and PBS soaked 5 minutes, the reuse volume fraction is the PBS diluent sealing of 5～10% normal goats serum, incubated at room 10 minutes, the deblocking liquid that inclines drips the immune serum of aforementioned acquisition again, hatched 1～2 hour for 37 ℃, PBS flushing, the biotin labeling that drips dilution again two anti-(with containing the PBS dilution that volume fraction is 1% bovine serum albumin) was hatched 10～30 minutes for 37 ℃, the PBS flushing, drip the Radix Cochleariae officinalis hydrogen peroxide enzyme labelling strepto-avidin (diluting) of dilution again, hatched 10～30 minutes for 37 ℃, the PBS flushing with PBS, the DAB colour developing, tap water fully washes, and redyes mounting, put microscopically and observe, film making.
The result as shown in Figure 3, wherein A is the blank group, and B is an experimental group, and visible CatSper1 has expression in testis tissue, it mainly is expressed in the round spermatid stage of testis tissue, and the anti-CatSper1 antibody that adopts vaccine immunity of the present invention to obtain can be effectively in conjunction with spermatid.
(2) immunofluorescence detects CatSper1
The purification sperm is evenly coated on the microscope slide, acetone fixed 15 minutes, after containing PBS (the being PBST) washing that volume fraction is 0.1% Triton-100, add peroxide enzyme inhibitor deactivating endogenous peroxydase again, after the PBS washing, put the wet box inner sealing of non-immune serum 10 minutes, and added the serum (dilution factor is 1: 300, is 1% hyclone dilution with volume fraction) of aforementioned acquisition again, 4 ℃ of overnight incubation, after the PBS washing, add marked by fluorescein isothiocyanate two again and resist, hatched under the room temperature 40 minutes, after the PBS washing, with volume fraction is 90% glycerol mounting, puts under the fluorescence microscope and observes, film making.
The result as shown in Figure 4, wherein A is the blank group, B is an experimental group, as seen adopts anti-CatSper1 antibodies that vaccine immunity of the present invention obtains on the principal piece and latter end of spermatic flagellum.
(3) ABT
The purification sperm is transferred in Biggers-Whitten-Whittingham (BWW) culture fluid, it is evenly distributed, density is about 1 * 10
6Individual/mL, add the anti-CatSper1 antibody that adopts vaccine immunity acquisition of the present invention and make the antibody final concentration be respectively 5 μ g/mL and 10 μ g/mL, each antibody concentration is set up 3 multiple holes, establish blank group (not adding anti-CatSper1 antibody) simultaneously, hatch for 37 ℃, calculate the motion sperm quantity with the area of computer aided sperm analysis system respectively after hatching 1,2,4 hour, each hole is with 3 visuals field of machine testing, experiment repeats 6 times, and data are carried out statistical analysis.
The result as shown in Figure 5, the anti-CatSper1 antibody incubation sperm that adopts vaccine immunity acquisition of the present invention is after 1,2,4 hours, motion sperm number average is than the obvious minimizing of blank group and become dose dependent, shows that the anti-CatSper1 antibody that adopts vaccine immunity of the present invention to obtain can suppress the motion of sperm.
Three, breeding paired experiment
Male BALB/c mouse is divided into experimental group, negative control group and blank group at random, 10 every group; Experimental group is an immunogen with CatSper1 gene vaccine of the present invention, negative control group is an immunogen with irrelevant contrast OVA gene vaccine (making with the preparation method of OVA full-length cDNA replaced C atSper1 full-length cDNA according to gene vaccine of the present invention), and the blank group is immunogen with PBS; Each organizes the dosage capable peritoneal immunity of mice by 100 μ L, every 1 week immunity 1 time, continuous immunity 3 times, last is after 1 week of immunity, female, male BALB/c mouse is mated copulation by 2: 1 female-male proportion of every cage, after 2 weeks female, male BALB/c mouse is separately raised separately, observe female Mus farrowing rate and litter size.
The result as shown in Figure 6 and Figure 7, the average farrowing rate of the female BALB/c mouse of experimental group is 23%, average litter size is 3.6; The average farrowing rate of the female BALB/c mouse of negative control group is 91%, and average litter size is 7.3; The average farrowing rate of the female BALB/c mouse of blank group is 93%, and average litter size is 6.9, shows that CatSper1 gene vaccine of the present invention can specificity reduces the average farrowing rate and the litter size of BALB/c mouse.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Sequence table
＜110〉Military Medical Univ No.3, P.L.A
＜120〉gene vaccine of sperm-specific cationic channel protein CatSper 1 and preparation method thereof
<160>6
<210>1
<211>2343
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉CatSper1 full-length cDNA
<400>1
atggatcaaa?actcagtgcc?tgaaaaggct?cagaatgagg?cagacaccaa?taacgcagat 60
aggttctttc?gctctcactc?atcaccccca?caccacaggc?caggccacag?cagagctctc 120
caccattacg?agttgcacca?tcacggcgtg?ccccaccaac?gtggtgaatc?tcaccaccct 180
ccggagttcc?aagacttcca?cgaccaagcc?ttgtcctccc?atgtccacca?atctcaccac 240
cacagcgagg?cacggaatca?cggcagagcc?catggcccca?caggctttgg?tctggctccc 300
tctcaaggcg?ccgtcccctc?ccaccgttcc?tacggtgagg?actaccatga?tgagctccaa 360
cgtgatggca?ggaggcatca?tgatgggtcc?caatacggtg?ggttccatca?gcagagtgac 420
tcccattacc?atagggggtc?tcaccatggc?agaccccaat?atctcggtga?gaatttatcc 480
cactattcct?ctggcgtgcc?ccaccacggt?gaggcttccc?accatggtgg?gtcctacctc 540
ccccatggac?ccaatcccta?cagtgagtcc?ttccaccaca?gcgaggcttc?ccaccttagc 600
gggctccaac?acgatgagtc?ccagcatcac?caagtccccc?accgtggctg?gccccaccat 660
caccaagtcc?accaccatgg?caggtcccgt?catcatgaag?cccaccagca?tggaaagtct 720
cctcatcacg?gagagaccat?ttcccctcat?tcctctgtgg?ggtcctacca?gcgtgggata 780
tctgactatc?acagcgagta?ccaccaaggt?gatcaccacc?ccagtgagta?ccaccatggc 840
gaccatcccc?accacacaca?gcaccactac?caccagaccc?accggcaccg?agactaccat 900
cagcaccaag?accaccacgg?cgcgtatcat?tccagttacc?tccatggcga?ctacgtccag 960
agcacttccc?aactctctat?cccacacaca?tcccggagcc?tgattcacga?tgcccccggc?1020
cctgctgctt?ctcgtacagg?agtcttcccc?tatcacgtag?cacacccacg?gggctcggct?1080
cacagcatga?ctcggtcctc?cagcacaatc?cgctcacgtg?tcacccagat?gtccaaaaaa?1140
gtccataccc?aggatatctc?caccaaacat?tcagaagact?ggggcaaaga?agaagggcaa?1200
tttcagaaac?gcaaaaccgg?ccggctccag?cggacccgca?agaagggaca?ctctaccaat?1260
ctcttccagt?ggctgtggga?aaagctaacc?ttcctcattc?agggcttccg?ggaaatgatc?1320
cggaacctga?cccaatcctt?ggcctttgaa?actttcatct?tcttcgttgt?ctgcctcaac?1380
accgtcatgc?tggtggccca?gaccttcgct?gaagtcgaga?tccggggcga?gtggtacttc?1440
atggccttgg?actccatatt?cttctgcatc?tacgtggtgg?aagccctgct?caagatcatc?1500
gccctgggcc?tctcgtactt?ctttgacttc?tggaacaatt?tggacttctt?cattatggcc?1560
atggccgtgc?tggacttctt?gctgatgcag?acccactcct?tcgccatcta?ccaccaaagc?1620
ctcttccgga?tcctcaaggt?cttcaagagc?ctgcgggccc?tgagggcaat?ccgggtcctg?1680
cggaggctca?gcttcctgac?cagcgtccag?gaagtgacag?ggaccctggg?ccagtccttg?1740
ccgtccatcg?cagccatcct?catcctcatg?tttacctgcc?tcttcctctt?ctccgcggtc?1800
ctccgggcac?tgttccgcaa?atctgacccc?aagcgcttcc?agaacatctt?caccaccatc?1860
ttcaccctct?tcaccttgct?cacgctggat?gactggtccc?tcatctacat?ggacagccgt?1920
gcccagggcg?cctggtacat?cattcccatc?ctcgtaattt?acatcatcat?ccagtacttc?1980
atcttcctca?acctggtgat?tactgtcctg?gtggatagct?tccagacggc?gctgttcaaa?2040
ggccttgaga?aagcgaagca?ggagagggcc?gcccggatcc?aagagaagct?gctggaagac?2100
tcactgacgg?agctcagagc?tgcagagccc?aaagaggtgg?cgagtgaagg?caccatgctg?2160
aagcggctca?tcgagaaaaa?gtttgggacc?atgactgaga?agcagcagga?gctcctgttc?2220
cattacctgc?agctggtggc?aagcgtggag?caggagcagc?agaagttccg?ctcccaggca?2280
gccgtcatcg?atgagattgt?ggacaccaca?tttgaggctg?gagaagagga?cttcaggaat?2340
tga 2343
<210>2
<211>858
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉BAFF full-length cDNA
<400>2
atggatgact?ccacagaaag?ggagcagtca?cgccttactt?cttgccttaa?gaaaagagaa 60
gaaatgaaac?tgaaggagtg?tgtttccatc?ctcccacgga?aggaaagccc?ctctgtccga 120
tcctccaaag?acggaaagct?gctggctgca?accttgctgc?tggcactgct?gtcttgctgc 180
ctcacggtgg?tgtctttcta?ccaggtggcc?gccctgcaag?gggacctggc?cagcctccgg 240
gcagagctgc?agggccacca?cgcggagaag?ctgccagcag?gagcaggagc?ccccaaggcc 300
ggcctggagg?aagctccagc?tgtcaccgcg?ggactgaaaa?tctttgaacc?accagctcca 360
ggagaaggca?actccagtca?gaacagcaga?aataagcgtg?ccgttcaggg?tccagaagaa 420
acagtcactc?aagactgctt?gcaactgatt?gcagacagtg?aaacaccaac?tatacaaaaa 480
ggatcttaca?catttgttcc?atggcttctc?agctttaaaa?ggggaagtgc?cctagaagaa 540
aaagagaata?aaatattggt?caaagaaact?ggttactttt?ttatatatgg?tcaggtttta 600
tatactgata?agacctacgc?catgggacat?ctaattcaga?ggaagaaggt?ccatgtcttt 660
ggggatgaat?tgagtctggt?gactttgttt?cgatgtattc?aaaatatgcc?tgaaacacta 720
cccaataatt?cctgctattc?agctggcatt?gcaaaactgg?aagaaggaga?tgaactccaa 780
cttgcaatac?caagagaaaa?tgcacaaata?tcactggatg?gagatgtcac?attttttggt 840
gcattgaaac?tgctgtga 858
<210>3
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward primer F1
<400>3
aatctgcaga?tggatcaaaa?ctcagtgcct 30
<210>4
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉downstream primer R1
<400>4
tacgaattct?caattcctga?agtcctcttc?tc 32
<210>5
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward primer F2
<400>5
gatcggatcc?atggatgact?ccacagaaag?g 31
<210>6
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉downstream primer R2
<400>6
acgcgtcgac?tcacagcagt?ttcaatgcac 30
Claims (5)
1. the gene vaccine of sperm-specific cationic channel protein CatSper 1 is characterized in that: the immunostimulating complex type vaccine that this vaccine is made up of CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector, saponin Quil A, cholesterol and lecithin.
2. according to the gene vaccine of the described sperm-specific cationic channel protein CatSper 1 of claim 1, it is characterized in that: described CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector is that the IRES upstream and downstream at the double gene coexpression eukaryotic vector that contains internal ribosome entry site IRES inserts CatSper1 full-length cDNA and BAFF full-length cDNA respectively.
3. according to the gene vaccine of the described sperm-specific cationic channel protein CatSper 1 of claim 1, it is characterized in that: the mass ratio of described CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector, saponin Quil A, cholesterol and lecithin is 5: 10: 1: 1.
4. the preparation method of the gene vaccine of the described sperm-specific cationic channel protein CatSper 1 of claim 1 is characterized in that: may further comprise the steps:
The structure of a, CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector: the IRES upstream of the CatSper1 full-length cDNA being inserted the double gene coexpression eukaryotic vector that contains IRES, again the BAFF full-length cDNA is inserted the IRES downstream of this eukaryotic vector, promptly get CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector;
The preparation of b, immunostimulating complex: is after PBS dissolves with CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector with phosphate buffer, add saponin Quil A, add cholesterol and lecithin again, make and contain CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector 0.5mg, saponin Quil A 1mg, cholesterol 0.1mg and lecithin 0.1mg in every ml soln, ice-bath ultrasonic is handled and is made abundant mixing of solution and emulsifying, reuse PBS dialysis, the little floccule of gained is the immunostimulating complex of formation, also is the gene vaccine of CatSper1.
5. according to the preparation method of the gene vaccine of the described sperm-specific cationic channel protein CatSper 1 of claim 4, it is characterized in that: the concrete grammar of described step a is:
A1, CatSper1 Full Length cDNA Cloning: extract people's mature sperm cell total rna, reverse transcription obtains total cDNA, be template with this total cDNA again, with sequence shown in SEQ ID No.3 and the SEQ ID No.4 is the upstream and downstream primer, and the pcr amplification two ends have the CatSper1 full-length cDNA of Pst I and EcoR I restriction enzyme site respectively, and the PCR condition is: 94 ℃ of pre-degeneration 3 minutes, 1 minute, 58 ℃ annealing of 94 ℃ of degeneration are 1 minute then, 72 ℃ were extended 1 minute, totally 30 circulations, and last 72 ℃ were extended 7 minutes; The purified rear clone of PCR product is gone into the pT-Easy carrier, gets recombinant vector pT-CatSper1;
A2, BAFF Full Length cDNA Cloning: extract the total RNA of people's spleen tissue, reverse transcription obtains total cDNA, be template with this total cDNA again, with sequence shown in SEQ ID No.5 and the SEQ ID No.6 is the upstream and downstream primer, the pcr amplification two ends have the B cell-stimulating factor full-length cDNA of BamH I and Sal I restriction enzyme site respectively, and the PCR condition is: 94 ℃ of pre-degeneration 5 minutes; 94 ℃ of degeneration are 50 seconds then, 58 ℃ of annealing 50 seconds, and 72 ℃ were extended totally 30 circulations 2 minutes; Last 72 ℃ were extended 10 minutes; The purified rear clone of PCR product is gone into the pT-Easy carrier, gets recombinant vector pT-BAFF;
The structure of a3, reorganization eukaryotic vector: in step a1 gained recombinant vector pT-CatSper1, go out the CatSper1 full-length cDNA with Pst I and EcoR I double digestion, purified back be connected with the eukaryotic vector pStar of EcoR I double digestion with Pst I equally, must recombinant vector pStar-CatSper1; In step b gained recombinant vector pT-BAFF, go out the BAFF full-length cDNA again with BamH I and Sal I double digestion, purified back is connected with same recombinant vector pStar-CatSper1 with BamH I and SalI double digestion, promptly gets CatSper1 and BAFF double gene coexpression reorganization eukaryotic vector pStar-CatSper1-IRES-BAFF.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101439643A CN101816799B (en) | 2010-04-09 | 2010-04-09 | Gene vaccine of sperm-specific cationic channel protein CatSper1 and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101439643A CN101816799B (en) | 2010-04-09 | 2010-04-09 | Gene vaccine of sperm-specific cationic channel protein CatSper1 and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101816799A true CN101816799A (en) | 2010-09-01 |
| CN101816799B CN101816799B (en) | 2011-12-07 |
Family
ID=42652170
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101439643A Expired - Fee Related CN101816799B (en) | 2010-04-09 | 2010-04-09 | Gene vaccine of sperm-specific cationic channel protein CatSper1 and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101816799B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103498001A (en) * | 2013-10-17 | 2014-01-08 | 于惠 | Real-time fluorescent PCR (polymerase chain reaction) test primer for BAFF (B-cell activating factor) gene and reagent kit |
| CN113454210A (en) * | 2019-02-08 | 2021-09-28 | 伦斯勒理工大学 | Contraceptive vaccine based on sperm-associated protein CATSPER |
-
2010
- 2010-04-09 CN CN2010101439643A patent/CN101816799B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 《INFECTION AND IMMUNITY》 20090413 Tertilt，C. et al. Expression of B-Cell Activating Factor Enhances Protective Immunity of a Vaccine against Pseudomonas aeruginosa 第3044页，摘要部分 1-3 第77卷, 第7期 2 * |
| 《中国优秀硕博士学位论文数据库（博士）医药卫生科技辑》 20030615 何畏 多价小鼠精子抗原表位肽的构建及其避孕效应研究 第54-58页 1-3 , 2 * |
| 《中国优秀硕博士学位论文数据库（硕士）医药卫生科技辑》 20070515 王伟 构建精子特异CATSPER1 DNA避孕疫苗的初步研究 1-3 , 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103498001A (en) * | 2013-10-17 | 2014-01-08 | 于惠 | Real-time fluorescent PCR (polymerase chain reaction) test primer for BAFF (B-cell activating factor) gene and reagent kit |
| CN103498001B (en) * | 2013-10-17 | 2015-09-02 | 南京迪安医学检验所有限公司 | BAFF gene real-time fluorescent PCR testing primer and test kit |
| CN113454210A (en) * | 2019-02-08 | 2021-09-28 | 伦斯勒理工大学 | Contraceptive vaccine based on sperm-associated protein CATSPER |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101816799B (en) | 2011-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Farris et al. | Vaccination against Chlamydia genital infection utilizing the murine C. muridarum model | |
| Wedlock et al. | Progress in the development of vaccines against rumen methanogens | |
| Zhao et al. | Vaccination of goats with DNA vaccines encoding H11 and IL-2 induces partial protection against Haemonchus contortus infection | |
| JP2010503384A (en) | Borrelia antigen | |
| Liu et al. | Toxoplasma gondii: immune response and protective efficacy induced by ROP16/GRA7 multicomponent DNA vaccine with a genetic adjuvant B7-2 | |
| Zhao et al. | Reducing microbial ureolytic activity in the rumen by immunization against urease therein | |
| Xu et al. | Intramuscular administration of a DNA vaccine encoding OmpK antigen induces humoral and cellular immune responses in flounder (Paralichthys olivaceus) and improves protection against Vibrio anguillarum | |
| Zhang et al. | The molecular characterization and immunity identification of Trichomonas vaginalis adhesion protein 33 (AP33) | |
| Czerkinsky et al. | Enteric vaccines for the developing world: a challenge for mucosal immunology | |
| CN101816799B (en) | Gene vaccine of sperm-specific cationic channel protein CatSper1 and preparation method thereof | |
| Sharma et al. | Formulation and delivery of vaccines: ongoing challenges for animal management | |
| Han et al. | Oral vaccination with inhibin DNA delivered using attenuated Salmonella choleraesuis for improving reproductive traits in mice | |
| Zhao et al. | PEDV-spike-protein-expressing mRNA vaccine protects piglets against PEDV challenge | |
| WO2008067651A1 (en) | Gender selection with the use of antibodies | |
| Bartolomé et al. | Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes as vehicles for DNA vaccines | |
| Aravind et al. | Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model | |
| CN101816798B (en) | Gene vaccine of sperm-specific protein Ropporin and preparation method thereof | |
| CN104673735B (en) | A kind of cortex chalone growth hormone release inhibiting hormone double expression DNA vaccination and preparation method and application | |
| BRPI0708959A2 (en) | brachyspira hyodysenteriae genes and proteins and use for diagnosis and therapy. | |
| Luo et al. | Immunogenicity study of plasmid DNA encoding mouse cysteine‐rich secretory protein‐1 (mCRISP 1) as a contraceptive vaccine | |
| Khulape et al. | Evaluation of a fusion gene-based DNA prime-protein boost vaccination strategy against Newcastle disease virus | |
| CN101897965B (en) | Hepatitis B virus dendritic cell therapeutic vaccine and preparation method thereof | |
| Xu et al. | Mucosal and cellular immune responses elicited by nasal and intramuscular inoculation with ASFV candidate immunogens | |
| CN103450353A (en) | Application of protein AIG_01780 in Rickettsia rickettsii resistant immune protection | |
| An et al. | In vitro and in vivo studies evaluating recombinant plasmid pCXN2‐mIzumo as a potential immunocontraceptive antigen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111207 Termination date: 20140409 |